A large proportion of biotechnology products, whether commercially available or in development, are protein therapeutics. There is hence a large and increasing demand for production of proteins in cell cultures, e.g., in animal cell cultures, and for improved methods related to such production. Thus, a significant quantity of research focuses on animal cell culture conditions and methods that can optimize the polypeptide output, i.e., conditions and methods that support high cell density and high titer of protein.
The C-terminal lysine variation is commonly observed in biopharmaceutical monoclonal antibodies. Monoclonal antibody heterogeneity can be attributed to various factors such as amino terminal modifications (e.g., to pyroglutamate), incomplete processing of the C-terminus, asparagine deamidation, phosphorylation, glycosylation, oxidation, mutations etc. These kinds of variations occur in many type of proteins and can impact their activity and stability in biotherapeutics. Homogeneity of antibodies is an important attribute and is regarded as essential for demonstrating safety and efficacy of drugs as required by FDA and other regulatory agencies.
Recombinant monoclonal antibodies have been shown to have C-terminal heterogeneity with either an arginine (Arg) or lysine (lys) at the C-terminus. When these C-terminal variants of MAbs are treated with Carboxypeptidase B, an exopeptidase, Arg and Lys are cleaved from the C-terminus of both the antibody subunits, eliminating C-terminal heterogeneity.
Carboxypeptidases (CPs) are enzymes that catalyze the hydrolysis of the C-terminal peptide bond in peptides and proteins. The removal of one or a few amino acids from the C-terminus of a peptide or protein can have profound effects on the biological activity of that molecule. Heterogeneity of MAb is common due to the various modifications introduced over the lifespan of the molecules from the point of synthesis to the point of complete clearance from the subject. It is important to study the modifications of a therapeutic as there is a possibility of it affecting the activity/safety of the preparation leading to loss of efficacy and risk of adverse side effects.
U.S. Pat. No. 5,126,250 discloses a method of reducing the heterogeneity of secreted antibodies from antibody-producing cells.